An APP mutation identified in an Italian family that results in an amino acid substitution at position 2 of amyloid ? (A?It) protects heterozygous carriers from AD even in advanced age. In vitro, A?It prevents wild-type A? (A?wt) from forming A? amyloid fibrils and oligomerization, suggesting that co-expression of A?It interferes with A? nucleation and/or polymerization, a mechanism that is hypothesized to inhibit A? oligomerization, amyloidogenesis and neurotoxicity in the heterozygous Italian APP (APPIt) carriers. In order to directly explore the in vivo effect of this mutation we have generated a transgenic mouse overexpressing human APP with the A673V Italian mutation under the control of Thy1.2 promoter sequences (TgAPPIt mice). We will investigate the anti-amyloidogenic effect of A?It when co-expressed with the human A?wt in vivo using amyloid-depositing mice (APP23) crossed with the TgAPPIt mice (Aim 1). Examining mechanism within the brain, we will determine whether A?It protection is due to the inhibition of A? nucleation or due to enhanced A? degradation, decreased oligomer/early aggregate stability, and/or enhanced clearance of the A?It/A?wt interacting peptides, testing the hypothesis that the amino-terminally mutated A?It interferes with the earliest events necessary for the initiation of ? amyloid-driven pathology. In an initial therapeutic strategy, the anti-amyloidogenic function of A?It will be investigated by administering to amyloid depositing mice exosomes-enriched extracellular vesicles (EV) as a source of A?It (Aim 2). In part because of their stability, autologous EV have many appealing properties as peptide, protein, and RNA delivery tools, and we have shown that brain EV contain APP, are highly enriched with APP carboxyl-terminal fragments (APP-CTFs) and are therefore a source of EV-generated A?. In preliminary studies we used our novel technique to isolate EV from the brain extracellular space of TgAPPIt mice and administered these intranasally to amyloid depositing mice. Amyloid deposition was reduced in mice treated with brain-derived EV from TgAPPIt mice compared to mice treated with EV isolated from the brain of wild-type mice, supporting our hypothesis that A?It interferes with developing A? pathology and supporting our idea that EV can be used to deliver protective constituents to the brain. TgAPPIt brain-derived EV will be delivered prior to amyloid deposition and at an age when amyloid deposition is abundant, and recipient mice will be examined for A? oligomerization, amyloid deposition, neuronal loss and behavioral deficits. We will further examine the anti-amyloidogenic potential and protective effects of A?It using EV loaded with APP-A673V mRNA or A?It peptides, as well as a non-fibrillogenic peptide comprised of the 6 amino- terminal residues of A?It. These studies are timely, given the growing interest in developing EV as therapeutic vehicles for both systemic and brain disorders, and our investigations of EV as candidates to act as vehicles to deliver protective A?It within the brain as a foundation for developing novel EV-based treatments for AD.